Buell Margaret, Chitwood James L, Ross Pablo J
Department of Animal Science, University of California, Davis, CA, United States.
Department of Animal Science, University of California, Davis, CA, United States.
Anim Reprod Sci. 2015 Mar;154:16-24. doi: 10.1016/j.anireprosci.2014.12.012. Epub 2014 Dec 26.
Removal of oocytes from their natural inhibitory follicular environment results in spontaneous resumption of meiosis independent of normal signaling events that occur in vivo. Controlling the onset of meiotic resumption via maintenance of elevated oocyte cAMP levels with adenylyl cyclase (AC) activation and phosphodiesterase (PDE) inhibition, and subsequent hormone stimulation with follicle FSH has been shown to dramatically improve developmental competence of bovine and murine IVM oocytes. This study evaluated the effect of cAMP modulation during IVM of sheep oocytes on meiotic progression and development to blastocyst after parthenogenetic activation. Changes in oocyte cAMP levels were quantified during the first 2h of in vitro maturation in control or cAMP-modulating medium. No significant changes in intra-oocyte cAMP were observed under control conditions, though a slight and transient drop was noticed at 15 min of maturation. Addition of the AC stimulator Forskolin and the PDE inhibitors IBMX altered the cAMP profile, resulting in 10-fold elevation of cAMP by 15 min and sustained >3-fold elevated levels from 30 to 120 min. The effect of cAMP elevation on meiotic resumption was measured by completion of germinal vesicle breakdown. Modulated oocytes were significantly delayed when compared to control media oocytes. Also, progression to MII was significantly delayed in modulated versus control oocytes at 20 and 24h, though no differences persisted to 28 h. Lastly, when control and modulated oocytes were parthenogenetically activated, no differences in blastocyst formation were observed. Thus, while cAMP modulation delayed meiotic progression, it did not improve developmental competence of sheep IVM oocytes.
将卵母细胞从其天然的抑制性卵泡环境中取出会导致减数分裂自发恢复，这与体内发生的正常信号事件无关。通过激活腺苷酸环化酶（AC）和抑制磷酸二酯酶（PDE）来维持卵母细胞cAMP水平升高，进而控制减数分裂恢复的起始，随后用卵泡刺激素（FSH）进行激素刺激，已被证明可显著提高牛和鼠体外成熟（IVM）卵母细胞的发育能力。本研究评估了绵羊卵母细胞IVM过程中cAMP调节对孤雌激活后减数分裂进程和发育至囊胚的影响。在对照或cAMP调节培养基中体外成熟的前2小时内，对卵母细胞cAMP水平的变化进行了定量分析。在对照条件下，未观察到卵母细胞内cAMP有显著变化，不过在成熟15分钟时注意到有轻微且短暂的下降。添加AC刺激剂福斯高林和PDE抑制剂异丁基甲基黄嘌呤改变了cAMP谱，导致15分钟时cAMP升高10倍，并在30至120分钟内持续保持>3倍的升高水平。通过生发泡破裂的完成情况来测量cAMP升高对减数分裂恢复的影响。与对照培养基中的卵母细胞相比，经调节的卵母细胞显著延迟。此外，在20小时和24小时时，经调节的卵母细胞与对照卵母细胞相比，向MII期的进展显著延迟，不过到28小时时差异不再存在。最后，当对照和经调节的卵母细胞进行孤雌激活时，未观察到囊胚形成有差异。因此，虽然cAMP调节延迟了减数分裂进程，但并未提高绵羊IVM卵母细胞的发育能力。
